15d-PGJ2-Loaded Solid Lipid Nanoparticles: Physicochemical ...

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RESEARCH ARTICLE 15d-PGJ 2 -Loaded Solid Lipid Nanoparticles: Physicochemical Characterization and Evaluation of Pharmacological Effects on Inflammation Nathalie Ferreira Silva de Melo 1,2, Cristina Gomes de Macedo 3, Ricardo Bonfante 3, Henrique Ballassini Abdalla 3, Camila Morais Gonçalves da Silva 4, Tatiane Pasquoto 5, Renata de Lima 5, Leonardo Fernandes Fraceto 2 , Juliana Trindade Clemente- Napimoga 3, Marcelo Henrique Napimoga 1* 1 Laboratory of Immunology and Molecular Biology, São Leopoldo Mandic Institute and Researcher Center, Campinas, Brazil, 2 Department of Environmental Engineering, São Paulo State University (UNESP), Sorocaba, Brazil, 3 Department of Physiological Sciences, Piracicaba Dental School, University of Campinas, Campinas, Brazil, 4 Department of Biochemistry, State University of Campinas (UNICAMP), Campinas, Brazil, 5 Department of Biotechnology, University of Sorocaba (UNISO), Sorocaba, Brazil These authors contributed equally to this work. These authors also contributed equally to this work. * [email protected] Abstract 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), a peroxisome proliferator-activated receptor- γ (PPAR-γ) agonist, has physiological properties including pronounced anti-inflammatory activity, though it binds strongly to serum albumin. The use of solid lipid nanoparticles (SLN) can improve therapeutic properties increasing drug efficiency and availability. 15d-PGJ 2 - SLN was therefore developed and investigated in terms of its immunomodulatory potential. 15d-PGJ 2 -SLN and unloaded SLN were physicochemically characterized and experiments in vivo were performed. Animals were pretreated with 15d-PGJ 2 -SLN at concentrations of 3, 10 or 30 μg kg -1 before inflammatory stimulus with carrageenan (Cg), lipopolysaccharide (LPS) or mBSA (immune response). Interleukins (IL-1β, IL-10 and IL-17) levels were also evaluated in exudates. The 15d-PGJ 2 -SLN system showed good colloidal parameters and encapsulation efficiency of 96%. The results showed that the formulation was stable for up to 120 days with low hemolytic effects. The 15d-PGJ 2 -SLN formulation was able to reduce neutrophil migration in three inflammation models tested using low concentrations of 15d- PGJ 2 . Additionally, 15d-PGJ 2 -SLN increased IL-10 levels and reduced IL-1β as well as IL-17 in peritoneal fluid. The new 15d-PGJ 2 -SLN formulation highlights perspectives of a potent anti-inflammatory system using low concentrations of 15d-PGJ 2 . PLOS ONE | DOI:10.1371/journal.pone.0161796 August 30, 2016 1 / 15 a11111 OPEN ACCESS Citation: de Melo NFS, de Macedo CG, Bonfante R, Abdalla HB, da Silva CMG, Pasquoto T, et al. (2016) 15d-PGJ 2 -Loaded Solid Lipid Nanoparticles: Physicochemical Characterization and Evaluation of Pharmacological Effects on Inflammation. PLoS ONE 11(8): e0161796. doi:10.1371/journal.pone.0161796 Editor: Muzamil Ahmad, Indian Institute of Integrative Medicine CSIR, INDIA Received: June 13, 2016 Accepted: August 11, 2016 Published: August 30, 2016 Copyright: © 2016 de Melo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: The authors gratefully acknowledge financial support from the Brazilian funding agencies São Paulo Research Foundation (FAPESP) and National Council for Scientific and Technological Development (CNPq). NFSM was supported by a research fellowship grant# 2014/11016-8, São Paulo Research Foundation. MHN was supported by a research fellowship grant # 303555/2013-0 (CNPq).

Transcript of 15d-PGJ2-Loaded Solid Lipid Nanoparticles: Physicochemical ...

RESEARCH ARTICLE

15d-PGJ2-Loaded Solid Lipid Nanoparticles:Physicochemical Characterization andEvaluation of Pharmacological Effects onInflammationNathalie Ferreira Silva de Melo1,2☯, Cristina Gomes de Macedo3☯, Ricardo Bonfante3☯,Henrique Ballassini Abdalla3☯, Camila Morais Gonçalves da Silva4‡, Tatiane Pasquoto5‡,Renata de Lima5‡, Leonardo Fernandes Fraceto2, Juliana Trindade Clemente-Napimoga3☯, Marcelo Henrique Napimoga1☯*

1 Laboratory of Immunology and Molecular Biology, São Leopoldo Mandic Institute and Researcher Center,Campinas, Brazil, 2 Department of Environmental Engineering, São Paulo State University (UNESP),Sorocaba, Brazil, 3 Department of Physiological Sciences, Piracicaba Dental School, University ofCampinas, Campinas, Brazil, 4 Department of Biochemistry, State University of Campinas (UNICAMP),Campinas, Brazil, 5 Department of Biotechnology, University of Sorocaba (UNISO), Sorocaba, Brazil

☯ These authors contributed equally to this work.‡ These authors also contributed equally to this work.*[email protected]

Abstract15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a peroxisome proliferator-activated receptor-

γ (PPAR-γ) agonist, has physiological properties including pronounced anti-inflammatory

activity, though it binds strongly to serum albumin. The use of solid lipid nanoparticles (SLN)

can improve therapeutic properties increasing drug efficiency and availability. 15d-PGJ2-

SLN was therefore developed and investigated in terms of its immunomodulatory potential.

15d-PGJ2-SLN and unloaded SLN were physicochemically characterized and experiments

in vivo were performed. Animals were pretreated with 15d-PGJ2-SLN at concentrations of 3,

10 or 30 μg�kg-1 before inflammatory stimulus with carrageenan (Cg), lipopolysaccharide

(LPS) or mBSA (immune response). Interleukins (IL-1β, IL-10 and IL-17) levels were also

evaluated in exudates. The 15d-PGJ2-SLN system showed good colloidal parameters and

encapsulation efficiency of 96%. The results showed that the formulation was stable for up

to 120 days with low hemolytic effects. The 15d-PGJ2-SLN formulation was able to reduce

neutrophil migration in three inflammation models tested using low concentrations of 15d-

PGJ2. Additionally, 15d-PGJ2-SLN increased IL-10 levels and reduced IL-1β as well as

IL-17 in peritoneal fluid. The new 15d-PGJ2-SLN formulation highlights perspectives of a

potent anti-inflammatory system using low concentrations of 15d-PGJ2.

PLOS ONE | DOI:10.1371/journal.pone.0161796 August 30, 2016 1 / 15

a11111

OPEN ACCESS

Citation: de Melo NFS, de Macedo CG, Bonfante R,Abdalla HB, da Silva CMG, Pasquoto T, et al. (2016)15d-PGJ2-Loaded Solid Lipid Nanoparticles:Physicochemical Characterization and Evaluation ofPharmacological Effects on Inflammation. PLoS ONE11(8): e0161796. doi:10.1371/journal.pone.0161796

Editor: Muzamil Ahmad, Indian Institute of IntegrativeMedicine CSIR, INDIA

Received: June 13, 2016

Accepted: August 11, 2016

Published: August 30, 2016

Copyright: © 2016 de Melo et al. This is an openaccess article distributed under the terms of theCreative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in anymedium, provided the original author and source arecredited.

Data Availability Statement: All relevant data arewithin the paper.

Funding: The authors gratefully acknowledgefinancial support from the Brazilian funding agenciesSão Paulo Research Foundation (FAPESP) andNational Council for Scientific and TechnologicalDevelopment (CNPq). NFSM was supported by aresearch fellowship grant# 2014/11016-8, São PauloResearch Foundation. MHN was supported by aresearch fellowship grant # 303555/2013-0 (CNPq).

IntroductionDrug delivery systems have been developed to prolong and improve drug action while reducingtoxicity, thus overcoming common problems such as poor solubility in water [1,2].

Solid lipid nanoparticles (SLN), submicron lipid carriers sized between 50 and 1000 nm, arecomposed of biocompatible materials able to incorporate mainly lipophilic drugs. SLN are consti-tuted by an external phase (an emulsifier and water) and an inner layer composed of lipid matrix,where the drug is dispersed [3–5]. Such nanocarriers feature low toxicity and cause no irritationto tissues, hence the growing interest in their use in the treatment of inflammatory diseases [6].

Inflammation is an organic response that precedes tissue injury or infection. This physiolog-ical process involves a coordinated action between the immune system and the damaged tissue,which stimulates infiltration and subsequent activation of inflammatory cells with release ofcytokines and other mediators [7]. In such scenario, 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2) stands out as a potential anti-inflammatory molecule. This kind of prostaglandin is aPPAR-γ (peroxisome proliferator-activated receptor-γ) agonist and it is derived from thecyclooxygenase pathway, participating in the resolution phase of acute inflammation [8–10].

Studies have shown that PPAR-γ agonists are being used in inflammatory disorders becausethey are able to regulate the immune response [9–14]. In a previous study, it was demonstrated that1 mg�kg-1 of plain 15d-PGJ2 is required to reduce neutrophil migration to an inflammation site [12]due to the high affinity of such molecule to serum proteins [8]. In order to increase the bioaviabilityand improve the pharmacological properties of 15d-PGJ2, our group demonstrated that PLGA-encapsulated 15d-PGJ2 achieved the same therapeutic effect as free 15d- PGJ2 in several inflamma-tion models at a concentration 33 times lower than the latter [15]. Encapsulated 15d-PGJ2 was alsoable to inhibit bone loss in periodontal disease secondary to reduced gingival inflammation [16].Such findings highlight the promising applications of 15d- PGJ2 encapsulated in nanoparticles.

The new formulation addressed in this study has advantages over other colloidal nanocarrierssuch as polymeric nanoparticles. Besides being made of physiologically well-tolerated materials,SLN have been approved for human use based on their low toxicity, reportedly 10 to 100 timeslower than polymeric nanoparticles. SLN also have good stability and wide range of administra-tion routes, including parenteral [3,5]. SLN are produced from lipids that are solid at room tem-perature such as mono-, di- or triglycerides, fatty acids or waxes. Such lipids are stabilized by oneor a mixture of emulsifiers to prevent nanoparticle agglomeration. Most such materials are cur-rently used in pharmaceutical or cosmetic formulations, which highlights their low toxicity [17].

SLN have shown to be better tolerated in vivo than polymeric nanoparticles, since the poly-mers used in the latter may carry an intrinsic element of cytotoxicity [18,19]. Moreover, thecomposition of the formulation used in this study, namely tripalmitin as lipid matrix and PVAas emulsifier, has been shown to be suitable as a sustained delivery system of bioactive mole-cules due to their good physicochemical characteristics such as size, zeta potential, high encap-sulation efficiency, etc [20,21].

There have only been a few reports on the association of 15d-PGJ2 to lipid systems [22],though none amounted to a study in vivo on the anti-inflammatory activity of such systems.The purpose of this study was therefore to address the physicochemical properties and thepotential anti-inflammatory activity of the novel 15d-PGJ2-SLN formulation in vivo.

Materials and Methods

Preparation of 15d-PGJ2-Loaded SLNThe nanoparticles were prepared via the emulsification/solvent evaporation method describedelsewhere [21,23], with some modifications. Firstly, the organic phase composed by the lipid

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Competing Interests: The authors have declaredthat no competing interests exist.

glyceryl tripalmitate (150 mg) and 15d-PGJ2 (100 μg) was dissolved in chloroform (5 mL). Theaqueous phase was composed by polyvinyl alcohol (1%, w/v) and deionized water (30 mL).The organic phase was added to the aqueous phase and this mixture was sonicated for 5 min at40 W on a probe sonicator yielding an emulsion, which in turn was mixed on an Ultra Turraxhomogenizer at 18,000 rpm for 7 min. The organic solvent was eliminated under low pressureusing a rotating evaporator and the final volume was 16 mL of 15d-PGJ2 at a concentration of6.25 μg.mL-1. A control formulation was also prepared without 15d-PGJ2.

Encapsulation Efficiency of 15d-PGJ2The amount of 15d-PGJ2 encapsulated by the SLN system was determined by ultrafiltration/centrifugation using Amicon1 ultrafiltration devices (10 kDa MWCO; Millipore1). 15d-PGJ2-SLN suspensions were centrifuged and the filtrate was analyzed using high performance liquidchromatography (HPLC). 15d-PGJ2 was quantified in a Varian ProStar equipment (Agilent1

Technologies), PS210 isocratic pump and a UV-Vis detector operating at 205 nm. A Gemini1

C18 column NX 5μ C18 110 Å, 150 x 4.6 mm (Phenomenex1) was used for the mobile phase ofsodium phosphate monobasic solution (pH 3.5; 0.01M) and acetonitrile (58:42, v/v) at 1 mL.min-1. The sample injection volume was 100 μL [15]. Encapsulation efficiency was then deter-mined from the difference between 15d-PGJ2 concentration measured in the ultrafiltrate andits total concentration (100%) in the nanoparticle suspension.

Size, Polydispersion, Zeta Potential and pH MeasurementsThe hydrodynamic diameter and polydispersion (PI) of nanoparticles were determinated usingdynamic light scattering and the zeta potential was evaluated by microeletrophoresis. A ZetaSi-zer Nano ZS 90 analyzer (Malvern1 Instruments) was used to perform the measurements(25°C, with an angle of 90°) with a dilution factor of 100 times. The pH values of the suspen-sions were evaluated using a calibrated pHmeter (Tecnal1, Brazil). The results were shown asthe means of five measures (mean ± SD). The physicochemical stability of the nanoparticleswas evaluated as a function of time, analyzing the suspension over 120 days [15,21,24].

Nanoparticle Tracking Analysis (NTA)SLN size distribution was investigated through NTA. The analyses were carried out on a Nano-Sight LM 10 instrument system, green laser beam (532 nm) and sCMOS camera, all controlledvia dedicated NanoSight v.2.3 software (Malvern1 Instruments, UK). Samples were diluted10,000 times and analyzed in triplicate injecting the sample (1 mL) into the cell. Five individualvideos of Brownian motion were recorded for each replicate at two thousand particles per repli-cate. Finally, the result achieved was the concentration of the particles as a function of size dis-tribution [20,21].

Determination of Nanoparticle MorphologyNanoparticle morphology was determined using transmission electron microscopy (TEM).SLN suspensions with or without the drug were diluted and applied to copper grids(200-mesh) coated with carbon film. The samples were dried at room temperature and werecontrasted using uranyl acetate (2%) and analyzed using a Zeiss LEO 906 microscope operatingat 80 kV, preserving the nanoparticle morphology [21,25].

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In Vitro Release AssayThe release profile of encapsulated 15d-PGJ2 in SLN was evaluated employing a donor-accep-tor compartments system, with an interposed cellulose membrane (MWCO 1 kDa). The sys-tem was maintained under constant magnetic stirring and sink conditions. Samples wereapplied to the surface of the donor compartment. From the acceptor compartment, aliquots of1 mL were collected over 6 hours. The concentration of the drug released was determined byHPLC in the acceptor compartment. The experiment was carried out in triplicate [16,24].

The release mechanism of SLN-encapsulated 15d-PGJ2 was evaluated using the Baker-Lons-dale theoretical model. This is based on the Higuchi model and is intended at elucidating drugrelease phenomena from spherical devices, such as micro and nanoparticles [26].

Evaluation of Cellular ViabilityCell viability experiment was established using the MTT reduction test. The experiment wasperformed on Balb-c 3T3 fibroblasts cultured in DMEM (supplemented with 10% fetal bovineserum, 1% penincilin and streptomycin sulfate). Briefly, cells were seeded into culture platesand incubated for 48 h. They were then exposed to free 15d-PGJ2 and SLN suspension (withand without 15d-PGJ2) at drug concentrations ranging from 0.06 to 2.2 μg.mL-1 for 24 hours.The cells were then incubated with MTT for 2 h at 37°C and purple formazan was quantifiedusing a plate reader at 570 nm. Cell viability was calculated as absorbance of converted dye[20,21,24].

AnimalsMale Balb/c mice (20–25 g) were used in this investigation. The animals were maintained in atemperature-controlled room (12:12 h light–dark cycle) and provided water and food ad libi-tum. This study was approved by the Ethics Committee on Animal Research of the Universityof Campinas (registration number 3623-1/2015) and all animals were manipulated in accor-dance with the Guiding Principles for the Care and Use of Animals.

Antigen-Induced PeritonitisThe immunization procedure occurred as previously described [15]. On day one, the animalswere exposed to the antigen via a subcutaneous (s.c.) injection of 100 μL of saline, 100 μL ofcomplete Freund’s adjuvant and 500 μg of methylated bovine serum albumin (mBSA) antigen.The animals were boosted on days 7 and 14 with mBSA dissolved in incomplete Freund’s adju-vant. Non-immunized (NI) animals received similar treatment, but without the antigen. Allanimals were treated with 15d-PGJ2-SLN at 3, 10 or 30 μg�kg-1 or empty SLN and challengedwith either mBSA (30 μg/cavity, intraperitoneal (i.p.)) or saline control on day 21. Four hoursafter the mBSA challenge, the animals were sacrificed by isoflurane inhalation and the perito-neal cavity was washed with 3 mL of phosphate buffered saline (PBS) containing 1 mM ethyle-nediamine tetraacetic acid (EDTA) and the exudate was recovered [15,27].

Carrageenan-Induced Peritonitis and LPS-Induced Neutrophil MigrationThirty minutes prior to the challenge, the animals were pretreated subcutaneously with 200 μLof saline or 15d-PGJ2-SLN (3, 10 or 30 μg�kg-1). Inflammation was caused by i.p. injection ofcarrageenan (Cg at 500 μg/cavity in 200 μL). A control group with empty SLN was includedaiming to verify whether the vehicle alone could affect neutrophil migration. Two hundredmicroliters of vehicle were injected s.c. and after 30 minutes the animals were challenged withsaline or Cg (i.p.). As previously described, 4 hours after the Cg challenge, the animals were

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sacrificed and the peritoneal cavity was washed with PBS containing 1 mM EDTA and the peri-toneal fluid recovered [15].

For LPS-induced neutrophil migration, the animals were pretreated s.c. with saline(200 μL), 15d-PGJ2-SLN (3, 10 or 30 μg�kg-1) or empty SLN. Thirty minutes later, LPS solutionwas injected (100 ng/cavity, i.p.). Four hours after the LPS challenge, all animals underwent thesame procedure described for antigen-induced and Cg-induced peritonitis [15].

Cell Counts and Cytokine MeasurementsA Neubauer chamber was used for total cell counts, with samples diluted in Turk’s solution.Differential cell counts were determined from cytocentrifuge monolayers stained with Wright-Giemsa (100 counted cells). The results were expressed as the number of neutrophils per cavity(means ± SD) [15].

IL-1β, IL-10 and IL-17 levels in the peritoneal fluid were determined using commercial kitsof enzyme-linked immunosorbent assay (ELISA) (R&D Systems, USA) at optical density of490 nm. From the standard curves, the results were expressed as pg�mL-1 of cytokine.

Hemolytic AssayThe hemolytic effect provoked by nanoparticle suspensions was evaluated using mouse eryth-rocytes (0.15% hematocrit). Hemolysis percentage was determined by the quantity of hemoglo-bin released from red blood cells. The samples were incubated with 15d-PGJ2-SLN suspensionsat concentrations ranging from 6.25 to 125 ng.mL-1 of the drug. Empty SLN were evaluated atthe same nanoparticle concentration (~1.60 X 1013 nanoparticles/mL) tested for loaded nano-particles. Erythrocytes were incubated at 37°C for 15 min and centrifuged at 1500x g for 3 min.Hemoglobin in the supernatant was detected at 412 nm wavelength. The data were expressedas hemolysis percentage [15,28].

Statistical AnalysisFive animals were used per group for the experiments in vivo. Data were reported asmeans ± SD. Different treatments were compared using ANOVA and Bonferroni’s t-test forunpaired values. For the cytotoxic assays, data were analyzed using ANOVA with Tukey’s posthoc test. Statistical significance was set at P<0.05.

ResultsBoth SLN suspensions (empty and 15d-PGJ2 loaded) were prepared and colloidal parametersevaluated. The nanoparticles were measured for hydrodynamic diameter, polydispersion, zetapotential and morphology. The rationale behind developing lipid nanoparticles includes therelatively low costs of the raw materials and production as well as the excellent physicochemicalstability inherent of such preparations. The colloidal parameters are described in Table 1.

The results showed that the parameters evaluated for the nano suspensions were similar tothose described for colloidal suspensions and that such parameters were not affected by encap-sulation of the active ingredient [21]. The polydispersion index values were below 0.2 for bothsuspensions, indicating a narrow distribution of particle diameter; a negative zeta potential wasfound for both nano suspensions. Encapsulation efficiency was up to 95%. This high value is acombination of the lipophilicity and affinity of the drug for the lipid core.

The physicochemical stability of particles is important data to define suitability of formula-tions. The parameters size, polydispersion, zeta potential, pH and encapsulation efficiencywere monitored over 120 days of storage in ambar glass flasks. The size of nanoparticles in

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both suspensions analyzed had such little variation that it may be regarded as constantthroughout the period of 120 days, indicating no aggregation of particles. The remainingparameters followed suit, indicating suitable colloidal stability of the systems [20,21,29].

TEMmicrographs obtained for the SLN and 15d-PGJ2-SLN preparations are shown in Fig 1.The nanoparticles presented a spherical shape with diameters in the range of 200–300 nm (Fig1A–1D). The presence of agglomerates was a technical artifact that occurred secondary to sampledrying during processing for TEM analysis. Particle sizes were consistent with both the polydis-persion index and the mean diameter values obtained with the DLS technique [21,29].

The NTA technique was used to characterize the SLN system. The results of nanoparticleconcentration are shown in Fig 2. For empty SLN, the concentration was 1.90 (± 0.35) × 1013

particles per mL with average diameter of 274.6 ± 93.5 nm. For 15d-PGJ2–SLN the concentra-tion was 1.60 (± 0.69) × 1013 particles per mL with average diameter of 194.1 ± 61.9 nm. BothDLS and NTA provided similar range of diameter values and particle concentration, withmonomodal size distributions. This was an indication of homogeneity of the system, which isdesirable in nanoparticulated formulations, as it reduces interferences with drug-release byincreasing stability.

The release profile of 15d-PGJ2 from SLN suspension was investigated (Fig 3). In thismodel, only free molecules of the drug were able to transpose the membrane, while the nano-particles were retained, which enabled the evaluation of the interaction between drug andnanoparticles. 15d-PGJ2 was quantified by HPLC in aliquots collected from the acceptor.

Fig 3 illustrates the release profile of 15d-PGJ2 from the SLN system. The time taken for50% release (t50%) was around 100 min. Evaluation of the release mechanism of 15d-PGJ2 fromthe SLN systems was performed using the Baker-Lonsdale´s equation. Linear regressionresulted in correlation coefficient (r) values of 0.982, and release constant (k) values of 0.322 x10−2 (± 0.001) min-1. Compared with the release profile of 15d-PGJ2 from PLGA nanocapsules,the best fit was achieved using the Higuchi´s model (r = 0.972; k = 0.048 min-1/2) and the mainrelease mechanism involved is based on the Fick's Law of diffusion [16]. In this study, weachieved compatibility with the Baker–Lonsdale model indicating that the mechanism involvedis also diffusion. With respect to k values, the release profile obtained from 15d-PGJ2-SLN waslower than that from PLGA nanocapsules. This could be explained by the superior interactionbetween 15d-PGJ2 and the tripalmitin used in the production of SLN [30], also demonstratedvia the encapsulation efficiency values (>95% for SLN and 77% for PLGA nanocapsules). Thishigh interaction caused slower release, showing that the SLN system was more suitable for the15d-PGJ2 molecule.

Cell viability after exposure to SLN suspensions was assessed using the MTT reduction test.The experiments were performed in 3T3 fibroblasts incubated with free 15d-PGJ2 and encap-sulated 15d-PGJ2 (0.06–2.2μg.mL-1). Controls using empty nanoparticles were also tested atthe same nanoparticle concentration (~ 1.60 x 1013 nanoparticles/mL).

It is known that some ionizable groups present in polymers, lipids and other components ofthe suspensions could be somewhat toxic due to their interaction with cell membranes [19,31].

Table 1. Colloidal parameters of both SLN and 15d-PGJ2-SLN suspensions. Values are expressed as means ±standard deviations.

Parameters SLN (1st day) 15d-PGJ2-SLN (1st day) SLN (120th day) 15d-PGJ2-SLN (120th day)

Mean diameter (nm) 252.3 ± 8.7 283.6 ± 8.1 260.2 ± 4.9 289.5 ± 2.3

Polydispersion 0.105 ± 0.013 0.109 ± 0.048 0.147 ± 0.024 0.113 ± 0.017

Zeta potential (mV) -23.6 ± 0.3 -24.8 ± 0.7 -21.9± 1.1 -17.8± 0.3

pH 4.43 ± 0.06 4.41 ± 0.04 5.11 ± 0.02 5.22 ± 0.01

Encapsulation efficiency (%) - 96.8 ± 1.7 - 95.1 ± 0.8

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The results (Fig 4) showed that SLN decreased cell viability to 70% at the highest concentrationtested. Exposure to free 15d-PGJ2 reduced cell viability to 30%, mainly at higher concentra-tions, while exposure to the 15d-PGJ2-SLN formulation significantly increased cell viability atprostaglandin concentrations ranging from 0.9 to 2.2 μg.mL-1 (P< 0.05).

Fig 1. Micrographs obtained through transmission electronmicroscopy. (a-b) SLN and 15d-PGJ2-SLNat 77.500x magnification; (c-d) SLN and 15d-PGJ2-SLN at magnification 215.000x magnification. The barsindicate image scales.

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I.p. administration of mBSA in immunized animals significantly increased neutrophilmigration in the peritoneal cavity compared to saline or to mBSA in NI animals. Pretreatmentwith encapsulated 15d-PGJ2 at three concentrations (3, 10 or 30 μg�kg-1) was able to inhibitneutrophil migration (3, 10 or 30 μg�kg-1) (Fig 5A). Cg and LPS injections significantly

Fig 2. Nanoparticle concentration as a function of particle size (nm). (a) SLN and (b) 15d-PGJ2-SLN performed at 25°C (n = 5).

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Fig 3. Cumulative release profile of 15d-PGJ2–loaded SLN in vitro in aqueous solution at 25°C (n = 3).

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increased neutrophil migration in vivo compared to saline. Similarly to mBSA, administrationof the 15d-PGJ2-SLN formulation sharply decreased neutrophil migration induced by eitherstimuli (Fig 5B and 5C) (P< 0.05). Empty SLN were also tested in three models to verifywhether the nanoparticles were able to alter immune responses. The results indicate no signifi-cant change in neutrophil migration (P> 0.05). Similar findings were previously reported for15d-PGJ2-loaded polymeric nanocapsules [15].

IL-10 release induced by 15d-PGJ2-SLN was investigated in order to evaluate a possible reg-ulatory effect on neutrophil migration. A dose-dependent increase in IL-10 levels was observedin the peritoneal fluid of mice tested with 15d-PGJ2-SLN and challenged with mBSA or Cg orLPS. For mice tested with saline or SLN, no increase in IL-10 levels were observed in either ofthe three inflammation models (Fig 6A–6C).

Fig 4. Viability of Balb-c 3T3 cells using the MTT assay after exposure to free 15d-PGJ2 and bothnanosuspensions (n = 12). Data are represented as percentage of viable cells. (*a) 15d-PGJ2-SLN versus15d-PGJ2 (ANOVA followed by Tukey test; P < 0.05).

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Fig 5. Anti-inflammatory effect of SLN and 15d-PGJ2-SLN assessed in different inflammatory models. The animals were tested using saline or 15d-PGJ2-SLN (3, 10 or 30 μg�kg−1) prior to challenging them with mBSA (30 μg/cavity) (A), Cg (500 μg/cavity) (B) or LPS (100 ng/cavity) (C). Neutrophilmigration was evaluated 4h later. Results are expressed as mean values (±SD) of 5 animals per group. Different letters indicate statistical significancebetween groups (ANOVA followed by Bonferroni's t-test; P < 0.05).

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Release of pro-inflammatory cytokines such as IL- 1β (Fig 6D–6F) and IL -17 (Fig 6G–6I)was also evaluated since reduction in neutrophil migration could accompany a diminishedrelease of chemotactic agents. A dose-dependent decrease in IL-1β and IL-17 levels wasobserved in the peritoneal exudate of mice tested with 15d-PGJ2-SLN and challenged withmBSA, Cg or LPS, whereas the mice tested with saline or SLN alone showed no decrease incytokine levels for the inflammation models assessed.

The hemolytic effect of both loaded and unloaded SLN were investigated using red blood cellsfrommice (Fig 7). Exposure of cells to both formulations demonstrated low toxicity, as illustratedby the hemolysis values obtained even with the highest concentration tested (125 ng.mL-1)(~18% for SLN and ~19% for 15d-PGJ2-SLN suspensions). Blood toxicity of the empty PLGAnanocapsules and those containing 15d-PGJ2 was tested employing a hemolysis assay. The resultsrevealed a low toxicity of such formulations even at the highest concentration (125 ng.mL-1),

Fig 6. Influence of 15d-PGJ2-SLN (3, 10 or 30 mg�kg-1) on IL-10, IL 17 and IL-1β levels in exudate. (A, D, G) mBSA (30 μg/cavity), (B, E, H) Cg (500 μg/cavity), (C, F, I) LPS (100 ng/cavity). Results are expressed as mean values (±SD) of 5 animals per group. Different letters indicate statistical significancebetween groups (ANOVA followed by Bonferroni's t-test; P < 0.05).

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with 40% and 35% hemolysis reported for the empty and loaded nanocapsules, respectively [15],while for the SLN suspensions (empty and loaded nanoparticles), 18.2% and 19.4%, respectively.The hemolytic effect was therefore lower for the SLN than for the PLGA nanocapsules, conse-quently blood-compatibility was higher for the former, though the difference between the formu-lations was not statistically significant.

DiscussionNanoencapsulation has gained great interest in the pharmaceutical field. Among the nanopar-ticles used to encapsulate drugs, SLN can be highlighted due to its high stability, small size, pro-tection of labile drugs as well a change in their release profile [32]. The SLN systems presentedgood colloidal stability, as well as high affinity to the lipid matrix. The SLN system was able tomodify drug release profile and to maintain cell viability compared to the free drug. 15d-PGJ2encapsulation reduced neutrophil migration in 3 different inflammatory models due in part toa decrease in IL-1β and IL-17 levels as well an increase in IL-10.

The parameters mean diameter and polydispersion index remained practically constant,with no aggregates, demonstrating the high stability of such systems over the study period.Other critical parameter for colloidal stability is zeta potential. This parameter reflects nano-particles surface charge. Some surfactants used in this formulation, such as PVA, however,have a steric stabilization mechanism. In this case, the surface charge measured cannot be usedas the main stability parameter [1,20]. Nonetheless, the zeta potential values were not affectedby encapsulation of the drug, which indicates that the drug is mostly contained within thenanoparticles. Such findings demonstrate that the physicochemical stability of the SLN suspen-sion is highly suitable for 15d-PGJ2 encapsulation. Similar results have been reported for otherSLN suspensions [20,21,29].

As demonstrated, 15d-PGJ2 encapsulation into SLN was highly efficient (>95%), confirm-ing the affinity of the drug to the lipid phase of the SLN. It has previously been reported thatthe encapsulation efficiency of PLGA nanocapsules was approximately 77% for 15d-PGJ2 [15].SLN has therefore been proved more suitable to encapsulate 15d-PGJ2, due probably to thegreater affinity or higher solubility of such drug in the lipids used in this study.

Fig 7. Hemolysis assay employing 0.15% hematocrit, pH 7.4 at 37°C for SLN and 15d-PGJ2-SLN suspensions (n = 6).

doi:10.1371/journal.pone.0161796.g007

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The release profile of the 15d-PGJ2-SLN formulation was evaluated using a two-compart-ment model. Because of the low solubility of 15d-PGJ2 in water, it was not possible to investi-gate the release profile of the free drug. Comparing with a previous report, the system 15d-PGJ2-SLN presented a lower release profile than the drug encapsulated in polymeric nanocap-sules [16], which is desirable and indicates a technical advantage of the latter over the former.

The cell viability pattern observed for unloaded SLN could be attributed to exposure of thecells to residual PVA present in the formulation or even to the influence of the nanoparticlecharge. A study has shown that tripalmitin SLN reduced cell viability to 30% and this could beattributed to the formation of aggregates and changes in nanoparticle structure leading toreduced of cell viability [33]. Our experimental data showed that high doses (0.9–2.2 μg.mL-1)of free 15d-PGJ2 resulted in lower cell viability than the 15d-PGJ2-loaded SLN (P<0.05). Suchprotective an effect may be explained by a reduced availability of the drug secondary to a highencapsulation efficiency and modified release profile obtained with the nanoparticles [19]. Onthe other hand, taking into account the in vivo data, the dose of 10 μg.kg-1 was effective in all 3inflammatory models. This concentration normalized by body size means that the final con-centration was approximately 0.3 μg. From both the in vitro and in vivo findings, one may con-clude that the effective dose used did not induce significant side effects. Thus, the resultsconfirmed that 15d-PGJ2 encapsulation into SLN resulted in reduced drug availability, therebyfavoring a higher percentage of viable cells.

The hemolytic effects of SLN suspensions were evaluated in mouse erythrocytes in order toinvestigate blood compatibility of such formulations. Solid lipid nanoparticles are regarded asbiocompatible and biodegradable systems. This may be related to their physicochemical prop-erties, lipid composition, etc [34]. The results herein demonstrated that both loaded andunloaded formulations caused low hemolysis (below 20%) indicating biocompatibility. Thehemolytic effect observed was dose-dependent and could also be attributed to exposure toresidual PVA. Despite both formulations being biocompatible, SLN was superior at delivering15d-PGJ2 in biological systems.

Comparing the present findings to those obtained with PLGA nanocapsules published pre-viously [15], it is evident that SLN performed better than the polymeric nanocapsules.

It has been described that high doses of exogenous 15d-PGJ2 are needed to exert pharmaco-logical effects [11,12] and encapsulation of the drug into polymeric nanocapsules was able toprotect the molecule and promote anti-inflammatory effects at low doses [15,16,35]. In thisinvestigation, our results showed that the 15d-PGJ2-SLN system had an anti-inflammatoryactivity. In all three inflammatory models tested, 15d-PGJ2-SLN inhibited leukocyte migrationto an inflammatory site and, most importantly, 15d-PGJ2-SLN at 10 μg.kg-1 was able to signifi-cantly decrease neutrophil migration and pro-inflammatory cytokine levels (IL-1β and IL-17)as well as to significantly raise IL-10 levels. Compared to previous data using free 15d-PGJ2(1000 μg.kg-1) [12], the current formulation (15d-PGJ2-SLN) lowered the dose needed toobtain an anti-inflammatory effect by 100 times. Additionally, compared to the previous for-mulation tested (PLGA nanocapsules) [15], for which the effective dose was 30 μg.kg-1, thisnew nanocarrier managed to further reduce the dose 3-fold, demonstrating that the SLN sys-tem was the most effective at releasing the drug at the site of inflammation. The effect of free15d-PGJ2 was not evaluated in this study because it has previously been established that thefree drug at such low concentration does not exert an anti-inflammatory effect [15].

The advantages of this new carrier are higher stability, lower preparation costs and higherefficiency at releasing 15d-PGJ2 in three animal inflammation models compared to PLGAnanocapsules, highlighting its potential as an alternative delivery method for 15d-PGJ2. Theresults obtained herein open new perspectives and represent a great step towards the treatmentof inflammatory diseases using a sustained 15d-PGJ2 releasing method from nanoparticles.

Characterization and In Vivo Evaluation of 15d-PGJ2 Loaded in SLN

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Author Contributions

Conceptualization:NFSM LFF JTCNMHN.

Formal analysis: NFSM CGMMHN JTCN.

Funding acquisition: NFSMMHN.

Methodology:NFSM CGM RB HBA CMGS TP RL.

Project administration:MHN.

Resources: NFSM CGM RL.

Supervision:MHN.

Validation: NFSM.

Visualization: NFSMMHN.

Writing – original draft: NFSMMHN.

Writing – review & editing:NFSMMHN JTCN.

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